Cu(II)-Mediated direct 18F-dehydrofluorination of phosphine oxides in high molar activity

Background The 18F/19F-isotope exchange method employing P(V)-centered prosthetic groups demonstrates advantages in addressing mild one-step aqueous 18F-labeling of peptides and proteins. However, the molar activity (Am) achieved through isotope exchange remains relatively low, unless employing a high initial activity of [18F]F−. To overcome this drawback, our work introduces a novel approach through a Cu-mediated direct 18F-dehydrofluorination of phosphine oxides. This method leverages the straightforward separation of the 18F-labeled product from the phosphine oxide precursors, aiming to primarily increase Am. Results Through a 19F-dehydrofluorination efficiency test, Cu(OAc)2 was identified as the optimal oxidative metal salt, exhibiting a remarkable 100% conversion within one hour. Leveraging the straightforward separation of phosphine oxide precursors and phosphinic fluoride products, the Am of an activated ester, [18F]4, sees an impressive nearly 15-fold increase compared to the 18F/19F-isotope exchange, with the same initial activity of [18F]F−. Furthermore, this Cu(II)-mediated 18F-dehydrofluorination approach demonstrates tolerance up to 20% solvent water content, which enables the practical radiosynthesis of 18F-labeled water-soluble molecules under non-drying conditions. Conclusions The direct 18F-dehydrofluorination of phosphine oxide prosthetic groups has been successfully accomplished, achieving a high Am via Cu(II)-mediated oxidative addition and reductive elimination. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-023-00234-y.


Background
With a favorable half-life of 109.8 min, widespread availability, and a low maximum positron energy of 634 keV, 18 F that is typically produced in the form of [ 18 F]F − with high initial A m , is indispensable for obtaining high-resolution PET images in clinical and research settings (Wängler et al. 2012;Miller et al. 2008;Littich et al. 2012;Coenen et al. 2010;Jacobson et al. 2015;Cai et al. 2008).Aqueous 18 F-labeling methods, capable of accommodating the excellent solubility of various hydrophilic substrates and [ 18 F]F − derived from [ 18 O]H 2 O, have the potential to significantly reduce substantial loss of activity and time during the drying and redissolving of [ 18 F]F − .The 18 F/ 19 F-isotope exchange method, relying on B/Si/P-centered prosthetic groups for 18 F-labeling, has demonstrated advantages in the one-step aqueous labeling of peptides and a select range of small-molecular tracers (Liu et al. 2014a;Hong et al. 2019;Schirrmacher et al. 2006).High molar activity 18 F-AMBF 3 -TATE (> 111 GBq/μmol) and 18 F-SiFAlin-TATE (60 ± 7 GBq/μmol) have been achieved by isotope exchange of 18 F-fluoride with a high starting activity (8-37 GBq) and successfully applied in clinical studies (Liu et al. 2014a;Ilhan et al. 2020).However, attaining such high A m is contingent upon a low precursor loads that may otherwise compromise the radiochemical yield (RCY), and the utilization of high initial-A m [ 18 F]F − generated in a system devoid of fluorinated materials (Berridge et al. 2009).
In 2005, a groundbreaking method for labeling the cholinesterase inhibitor Dimefox (N,N,N' ,N'-tetramethylphosphorodiamidic fluoride) showcased the feasibility of constructing P-18 F bonds (Studenov et al. 2005).Notably, the initial approach utilized the labeling precursor N,N,N' ,N'-tetramethylphosphorodiamidic chloride, which exhibited poor stability and susceptibility to hydrolysis upon contact with water.Recognizing these drawbacks, we turn our attention to phosphine oxides-an extensively available class of pentavalent phosphine reagents known for their high solubility and stability in aqueous media.These phosphine oxides emerge as promising 18 F-labeling precursors, displaying an adequate polarity shift after fluorination that facilitates effective separation.Traditionally, the synthesis of organophosphorus fluorides from phosphine oxides involved one-pot-two-step methods, including nucleophilic attacks by F − on intermediates featuring leaving groups such as Cl (Gupta et al. 2008a, b;Bornemann et al. 2021;Purohit et al. 2015), alkyl sulfide (Timperley et al. 2005), phenyl ether (Wang et al. 2021), imidazole (Mou et al. 2021), or oxidative coupling between phosphine hydrogen oxide and NaF (Liu et al. 2014b) (Scheme 1a).Unfortunately, these approaches often necessitated highly toxic, corrosive chlorinating reagents or strong oxidants, making them less amenable to mild 18 F-labeling in an aqueous phase.An alternative avenue lies in the use of less toxic oxidative simple metal salts, which possess hydrates and are resilient to solvent effects.This approach presents a viable option for the direct dehydrofluorination of phosphine oxides through oxidative addition and reductive elimination.
In this study, a metal-mediated method for the direct radiofluorination of phosphine oxide prosthetic groups is developed (Scheme 1b).Initially, an array of metal salts including Cu(II), Cu(I), Ag(I), Pd(II), Fe(III), Fe(II), Ni(II), Mn(II), Zn(II), and Pt(II) undergoes investigation via a 19 F-fluorination efficiency test monitored by 19 F nuclear magnetic resonance (NMR).Subsequent optimization of the 19 F-dehydrofluorination reaction conditions is conducted, exploring variations in metal salt equivalents, solvents, fluorine sources, and reaction duration.Building upon these findings, the optimization of conditions for the oxidative metal-mediated 18 F-radiofluorination is pursued, with an additional focus on investigating water resistance properties.Capitalizing on the potential ease of separating the 18 F-labeled products from the precursors, the achieved A m via this direct 18 F-dehydrofluorination approach is compared with that obtained through isotope exchange with the same initial activity of [ 18 F]F − .The proposed mechanism involving oxidative addition and reductive elimination for this direct dehydrofluorination approach is observed through a 19 F-fluorination conversion test, monitored by 31 P NMR, incorporating free radical scavengers, organic acids, or organic bases as additives.

Synthesis
Phosphine oxide substrates 1a and 2a were obtained in yields of 97-98% through the reciprocal isomerization of the hydrolysis products of the corresponding chlorophosphane, as illustrated in Scheme 2a (Smoll et al. 2017).Compound 3a was procured directly from Energy Chemical Co., Ltd.(China), while 4a was synthesized through three distinct routes, as depicted in Scheme 2c, with yields ranging from 3% to 33%.The fluorination products, referred to as the reference compounds-phosphinic fluorides 1-4were prepared from phosphine oxides 1a-4a, employing CsF as the fluoride source in the presence of CuCl 2 , as outlined in Scheme 2b (Purohit et al. 2015).All synthesized compounds were characterized by nuclear magnetic resonance spectroscopy ( 1 H NMR,  (2024) 9:4 of these compounds were determined to be > 95% using thin-layer chromatography (TLC), NMR, and high-performance liquid chromatography (HPLC) methods.

Preliminary screening of oxidative metal salts
To assess the stability of phosphine oxides, di-tert-butylphosphine oxide 1a was dissolved in a mixture of water and acetonitrile at varying pH levels (1,4,7,10,12) and incubated at room temperature for 24 h.Remarkably, 1a demonstrated robust stability under all tested conditions, as depicted in Fig. 1a.Subsequently, employing compound 1a as the model substrate and tetrabutylammonium fluoride (TBAF) as the fluorination reagent, a comprehensive screening of oxidative metal salts, including Cu(II), Cu(I), Ag(I), Pd(II), Fe(III), Fe(II), Ni(II), Mn(II), Zn(II), and Pt(II) salts, were conducted under a general reaction formula outlined in Additional file 1: Scheme S1.The results indicated that, in addition to CuCl 2 , which has been reported in the literature to effectively mediate dehydrofluorination (Purohit et al. 2015), AgNO 3 and Cu(OAc) 2 (even achieving 100% dehydrofluorination conversion) exhibited outstanding fluorination efficiency (Fig. 1b and Additional file 1: Tables S1-S4).Conversely, dehydrofluorination conversions mediated by other transition metal salts were below 10%, with Pt(II) and Zn(II) metal salts exhibiting 0% dehydrofluorination conversion.Subsequently, the fluorination methods mediated by three metal salts-CuCl 2 , AgNO 3 , and Cu(OAc) 2 -were further optimized with respect to reaction time, reaction solvent, and fluorine source (Fig. 1c-e and Additional file 1: Tables S5-S7).Despite all three salts achieving fluorination yields exceeding 97% after optimization, Cu(OAc) 2 demonstrated complete conversion in less than one hour, indicating significantly higher fluorination efficiency than the other two metal salts (Fig. 1f and Additional file 1: Tables S8-S10).The water resistance of this fluorination method was also preliminary tested to provide insights for subsequent radiofluorination studies in aqueous media (Fig. 1g, h).Remarkably, a fluorination conversion of 70% was maintained when water was added in 10 equivalents of the substrate.
Even with a stoichiometric ratio of water to substrate increased to 100:1, a fluorination conversion of 7% was still observed in the 31 P NMR spectrum.

Radiochemistry
The optimization of 18 F-labeling conditions for this Cu(II)-mediated radiofluorination method was conducted using the activated ester substrate 4a, a promising 18 F-synthon, as the model substrate (Additional file 1: Scheme S3).Notably, the choice of solvent significantly influenced the radiochemistry conversions (RCCs), with the RCC of [ 18 F]4 reaching 49% when using the highly polar solvent DMSO, in which Cu(OAc) 2 is well solubilized (Fig. 2a).A time-RCC study revealed a rapid increase in RCC within the first 5 min, followed by a slight elevation over time, suggesting 10 min as an optimal reaction time considering decay effects (Fig. 2b).Temperature modulation proved to be crucial, with an unfavorable increase causing the reaction system to transition from blue to black.This transformation is assumed to involve the conversion of copper ions to copper oxides, resulting in a decline in RCC.Therefore, the optimal reaction temperature was determined to be 25 °C (Fig. 2c).The optimal amount of precursor was identified as 3 μmol, and the ideal amount of Cu(OAc) 2 was twice the precursor equivalent dissolved in 200 μL of solvent (Fig. 2d, e).Exploring the effects of different metal ions and phase transfer catalysts on 18 F-fluorination, it was determined that the most suitable fluorine source was [ 18 F]TBAF (Fig. 2f ).
A m of Cu(II)-mediated 18 F-dehydrofluorination versus isotope exchange Cu(OAc) 2 (2 equiv.)and precursor 4a (3 μmol) were individually dissolved in 100 μL DMSO and then sequentially added to the glass vial with dried [ 18 F]TBAF (20 mCi).The mixture was incubated at 25 °C for 10 min.Following purification by HPLC, the final product [ 18 F]4 was obtained in a 35% RCY, boasting a radiochemical purity of > 97% and a molar activity of 7.3 GBq/μmol.Similarly, precursor 4 (3 μmol) was dissolved in 200 μL CH 3 CN and added to the glass vial with dried [ 18 F]KF/K 222 (20 mCi).The mixture was incubated at 25 °C for 10 min.Post-purification by Sep-Pak C18 light cartridge, [ 18 F]4 was obtained in an 86% RCY, demonstrating a radiochemical purity of > 97% and a molar activity of 0.5 GBq/μmol.Notably, this work led to a 15-fold improvement in the A m of [ 18 F]4 compared to the isotope exchange method (Fig. 2i).

Mechanism of Cu(II)-mediated dehydrofluorination
In order to gain insight into the mechanism of Cu(OAc) 2 -mediated fluorination, we conducted a preliminary mechanistic study outlined in Scheme 3a.Previous studies have established that copper salts can directly react with Ph 2 P(O)H to generate phosphorus radicals (Ke et al. 2015;Yi et al. 2016;Yang et al. 2015).Thus, we hypothesized that this fluorination mechanism might involve a radical pathway.To test this hypothesis, radical trapping experiments were performed using the commonly employed radical capture agents, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 2,6-di-tert-butyl-4-methylphenol (BHT), known for effectively capturing free radicals in chemical reactions (Li et al. 2023;Ye et al. 2023).Surprisingly, the conversion of product 1 remained at 100% when 3.0 equivalents of TEMPO or BHT were added under standard condition.Furthermore, high-resolution mass spectrometry (HRMS) didn't detect TEMPOand BHT-coupled products.These results strongly suggest that this transformation may not actually involve the generation of radicals.Additionally, the addition of AcOH (2.0 equiv.) to the reaction under standard condition led to a significant decrease in the conversion of 1 from 100% to 38%.In contrast, the conversion of 1 remained essentially unchanged when 2.0 equivalents of base (TEA, Py, and DBU) were added.These results indicate that the pH of the reaction system may be a crucial factor influencing the reaction.We also speculated that AcOH was generated during the reaction, and the addition of AcOH inhibited the reaction, while the addition of these bases could react with the generated AcOH, promoting the reaction to proceed forward.
Based on the experimental results and literature research (Zhou et al. 2014;Shen et al. 2021), we propose a plausible mechanism for the Cu(OAc) 2 -mediated fluorination reaction, as illustrated in Scheme 3b.Initially, phosphine oxides undergo reciprocal isomerization from the pentavalent phosphorus compound A to the less-stable trivalent phosphorus compound B, exposing a pair of electrons.Subsequently, the coordination of copper fluoride with electrons on the trivalent phosphorus compound leads to the formation of the oxidation addition intermediate C. Finally, the reductive elimination process involves AcO − attacking the H atom of OH and the F atom attacking the P atom, resulting in the formation of the fluorinated products D, along with CuF and AcOH.

Discussion
Considering the established in vivo stable structures of phosphinic fluorides, compounds 1-3 were synthesized with a thoughtful consideration of both the site-blocking factor and the conjugation effect.To evaluate their hydrolytic stabilities, assessments were conducted in a mixed solvent containing D 2 O and CD 3 CN using 19 F NMR (Additional file 1: Figure S2).The site-blocking effect emerged as a crucial factor in stabilizing disubstituted phosphinic fluorides.Compound 1, characterized by a bis-tert-butyl structure with substantial steric hindrance, demonstrated no defluorination even after 9 days of incubation, aligning with findings from previous literature.In contrast, the diphenylsubstituted phosphoryl fluoride structure exhibited a higher susceptibility to hydrolysis.The stable phosphine oxides used in this method is, in fact, one of the reactants involved in the synthesis of phosphinic fluorides (the precursors for isotope exchange), which streamlines the synthesis pathway for the precursor.
The screening process involved the evaluation of various readily available oxidative metal salts, encompassing conventional halides, oxides, inorganic acid-derived metal salts, and organic acid-derived metal salts.While Cu(OAc) 2 demonstrated the highest fluorination efficiency, it's worth mentioning that the less toxic alternative AgNO 3 also exhibited satisfactory fluorination efficiency.While Cu(I)-based chelates are commonly used in clinical studies with various radiotracers (Anderson et al. 2009), it is crucial to consider the potential toxicity of Cu(OAc) 2 , even though minute amounts are used in PET imaging radiotracers.In order to ensure the removal of any Cu 2+ residue, inductively coupled plasma mass spectrometry (ICP-MS) analysis can be conducted after the HPLC purification.
The significant steric hindrance effect was observed to be unfavorable for nucleophilic attack by F − , as evidenced by the gradual increase in RCCs as the site resistance decreased.Therefore, achieving a delicate balance between hydrolytic stability and RCC (reactivity towards F − ), the tert-butylphenylphosphorylfluoride structure represents a reasonable compromise.
In the context of labeling hydrophilic substrates such as small molecules, polypeptides, and proteins, considering the water resistance property of the 18 F-labeling method is essential.Our method exhibits a remarkable tolerance of up to 20% solvent water content, rendering it suitable for the 18 F-labeling of certain water-soluble biomolecules and precursors that may be highly sensitive or insoluble in organic solvents.
The A m is a critical consideration in the preparation of a receptor-targeting tracer.This method significantly enhances the A m of the activated ester [ 18 F]4 by nearly 15-fold compared to the 18 F/ 19 F-isotope exchange method with the same precursor load and initial activity.
A review of previous studies revealed that the copper-mediated radiofluorination method plays a crucial role in the construction of aromatic C-18 F bonds, significantly broadening the chemical space of 18 F-labeling methodology, as well as improving the synthesis of radiopharmaceuticals and advancing PET imaging studies (Wright et al. 2020).In this work, we present a novel Cu(II)-mediated dehydrofluorination of phosphine oxides to construct P-18 F bonds.This method offers a possibility for 18 F-labeling of phosphorus-containing biomolecules and will hopefully be applied to the study of C-18 F bond formation as well as to simplify the process of probe production.

Conclusion
In summary, this work has disclosed a Cu(OAc) 2 -mediated 18 F-dehydrofluorination method for phosphine oxides, facilitating the formation of P-F bonds, and proposes a plausible mechanism for this process.In particular, the A m of the activated ester [ 18 F]4 is significantly increased by nearly 15-fold compared to the 18 F/ 19 F-isotope exchange method.Furthermore, the approach exhibits remarkable tolerance of up to 20% aqueous phase, holding promise for the realization of 18 F-labeled water-soluble biomolecules and drug molecules suitable for positron emission tomography imaging under non-drying conditions.Eventually, a Cu(II)-mediated redox fluorination mechanism was proposed by monitoring the change in conversion rate upon the addition of radical trappers and additives.
Proton-1, carbon-13, fluorine-19, and phosphorus-31 nuclear magnetic resonance ( 1 H, 13 C, 19 F, 31 P NMR) spectra were recorded on an AS 400 MHz NMR spectrometer (ZhongKeNiuJin, China, 1 H NMR at 400 MHz, 13 C NMR at 101 MHz, 19 F NMR at 376 MHz, 31 P NMR at 162 MHz).Tetramethylsilane (TMS) was used as an internal standard for 1 H NMR, and all the chemical shifts were reported as δ values relative to the internal TMS.Chemical shifts for protons were reported in parts per million (ppm) downfield from TMS and were referenced to residual protium in the solvent ( 1 H NMR: CDCl 3 at 7.26 ppm, D 2 O at 4.79 ppm, MeOD at 3.31 ppm, and DMSO-d 6 at 2.50 ppm).Chemical shifts for 13 C signals were referenced to the carbon resonances of the solvent peak ( 13 C NMR: CDCl 3 at 77.16 ppm, MeOD at 49.00 ppm, and DMSO-d 6 at 39.52 ppm).Multiplicity was defined by s (singlet), d (doublet), t (triplet), and m (multiplet).The coupling constants (J) were reported in Hertz (Hz).
HPLC separation was achieved on an Ultimate XB-C 18 (5 µm, 10 mm × 250 mm) column (Welch, China).HPLC analysis was achieved on a 5 C 18 -MS-II (4.4 µm, 4.6 mm × 250 mm) column (Nacalai Tesque Cosmosil, Japan).Thin layer chromatography (TLC) was performed on TLC Silica gel 60 F254 aluminum sheets (Energy, China), and visualized with short wave UV light (254 nm) or iodine staining.Sep-Pak ® light QMA cartridge (Waters, USA) was flushed with 5.0 mL KHCO 3 solution (0.5 mol L −1 ), air, 10.0 mL water and air before use.Sep-Pak ® Plus C18 cartridges (Waters, USA) was flushed with 5.0 mL alcohol, air, 10.0 mL water and air before use.RCCs were obtained by calculating the ratio of the radioactive peak area of a 18 F-product to the total radioactive peak area.RCCs were determined by radio-TLC and radio-HPLC.Radio-TLC was performed on a Mini-Scan (Eckert & Ziegler, Germany) equipped with a Flow-Count (Bioscan, USA).Radio-HPLC was performed on a Dionex Ulti-Mate 3000 HPLC (Thermo Fisher, USA) equipped with a SPD-20A UV detector (Thermo Fisher, USA) and a Gabi Star γ-radiation detector (Elysia Raytest, Hungary).

Benzyl 2-(tert-butylhydrophosphoryl)-2-methylpropanoate (4c)
Route 1: A 250 mL three-necked round bottom flask containing zinc powder (719.18mg, 11.00 mmol) was degassed and flushed with dry nitrogen and charged with 30 mL THF.A solution of iodine in 10 mL dry THF was added in the flask with vigorous stir.Next, a solution of benzyl 2-bromo-2-methylpropanoate (4d, 2.57 g, 10.00 mmol) in 40 mL THF was placed in dropping funnel and 5 mL of the solution was added to trigger the formation of Grignard reagent.After the mixture turning murky, residual 4d was added dropwise into the mixture for another 4 h.
After zinc powder was nearly consumed, tert-butyldichlorophosphane (TBDCP, 1.59 g, 10.00 mmol) in 40 mL THF was added dropwise into the flask under nitrogen in an icewater bath at 0 °C for 1 h.Then, allow the reaction mixture warm to room temperature Tang et al. EJNMMI Radiopharmacy and Chemistry (2024) 9:4 and was stirred for another 12 h.The reaction was then quenched with 1 mol•L −1 HCl (20 mL) and extracted with ethyl acetate (80 × 3 mL).The organic layers were combined and concentrated and washed with saturated brine (50 × 3 mL).The organic layer was then dried on magnesium sulfate, filtered, and concentrated again in vacuo.The concentrate was purified by a short column chromatography on silica gel using dichloromethane and methanol (80:1) as elute to give the corresponding white solid (37% yield).Route 2: A 100 mL three-necked round bottom flask containing Magnesium (Mg) chips (160.41 mg, 6.60 mmol) was degassed and flushed with dry nitrogen and charged with 20 mL THF.After heating the mixture at boiling point, a solution of benzyl 2-bromo-2-methylpropanoate (4d, 1.54 g, 6.00 mmol) in 20 mL THF was placed in dropping funnel and 4 mL of the solution was added to trigger the formation of Grignard reagent.After naturally cooling the flask to room temperature, the residual 4d was added dropwise into the mixture for another 4 h.
Route 2: A 100 mL round bottom flask was filled with ethyl 2-(tertbutylhydrophosphoryl)-2-methylpropanoate (4f, 1.10 g, 5.00 mmol), then a solution of sodium hydroxide (NaOH, 599.9 mg, 15 mmol) in 40 mL water was added to the reaction flask.After stirring for 5 h, the starting material was completely consumed (by TLC).Next, the mixture was acidified to pH = 2 with 1 mol•L −1 HCl and extracted with ethyl acetate (40 × 3 mL).The organic layer was then dried with magnesium sulfate, filtered, and concentrated in vacuo to give a white solid of pure compound 4b (91% yield).

Ethyl 2-(tert-butylhydrophosphoryl)-2-methylpropanoate (4f)
A 250 mL three-necked round bottom flask containing zinc powder (1.44 g, 22.00 mmol) was degassed and flushed with dry nitrogen and charged with 50 mL THF.A solution of iodine in 10 mL dry THF was added in the flask with vigorous stir.Next, a solution of ethyl 2-bromo-2-methylpropanoate (EBMPA, 3.90 g, 20.00 mmol) in 40 mL THF was placed in dropping funnel and 5 mL of the solution was added to trigger the formation of Grignard reagent.After the mixture turning loose and murky, residual 2-bromo-2-methylpropanoate was added dropwise into the mixture for another 4 h.

Screening of metal salts and optimization of Cu(OAc) 2 , CuCl 2 and AgNO 3 -mediated fluorination reaction conditions
Compound 1a (0.0100 g, 0.0617 mmol), metal salt (2 equiv.)and were added to a 2 mL centrifuge tube with solvent (0.5-0.6 mL) as the reaction solvent.Oxidative transition Tang et al.EJNMMI Radiopharmacy and Chemistry (2024) 9:4 metal salts, such as, Cu(II), Cu(I), Ag(I), Pd(II), Fe(III), Fe(II), Ni(II), Mn(II), Zn(II), and Pt(II) salts were tried.Each reactant was sonicated to dissolve as much as possible and then transferred to a magnetic stirrer.Then, fluorine source (2 equiv.) was added to the system, and the reaction was carried out at the corresponding temperature for 12 h.The reaction was quenched by adding saturated K 2 CO 3 solution to the reaction solution and centrifuged, and the supernatant was aspirated for 31 P NMR analysis (Additional file 1: Table S1-S7 and Figure S3).

Mechanism study
Compound 1a (0.0100 g, 0.0617 mmol), Cu(OAc) 2 (0.1233 mmol), TBAF (1 mol•L −1 in THF, 0.1233 mL, 0.1233 mmol), and additives (TEMPO, BHT, AcOH, TEA, Py or DBU, 2-3 equiv.), were added to a 2 mL tube with acetone (0.5 mL) as solvent and transferred to a magnetic stirrer.Then, TBAF (1 mol•L −1 in THF, 0.1233 mL, 0.1233 mmol) was added to the mixture and stirred at room temperature for 12 h.When the reaction was finished, the resulting mixture was extracted with saturated potassium carbonate solution, and the supernatant was collected for 31 P NMR analysis.The conversion of the compound 1 was shown in Additional file 1: Table S11.

H 2 O-Resistance of fluorination
Compound 1a (0.0100 g, 0.0617 mmol), Cu(OAc) 2 (0.0246 g, 0.1233 mmol), pure water according to the corresponding stoichiometric ratio and KF (0.0072 g, 0.1233 mmol) were sequentially added to a tube with DMSO (0.4 mL) as reaction solvent (Additional file 1: Scheme S2).The resulting mixture was stirred at room temperature.The reaction was quenched by adding saturated K 2 CO 3 solution to the reaction solution and centrifuged, and the supernatant was aspirated for 31 P NMR analysis (Additional file 1: Table S12, S13, and Figure S4).

The preparation of dried [ 18 F]KF
The [ 18 F]F − aqueous was taken in a clean glass vial and KOAc solution (KOAc: 0.5 mg, 5.0 μmol; 200 μL H 2 O) was added to disperse [ 18 F]F − uniformly at the bottom of the vial.The solution was azeotropically dried for three times (300 μL anhydrous CH 3 CN × 3) at 100 °C with nitrogen flow.Then the reaction vial was capped to obtain dried [ 18 F]KF for radiolabeling.

The preparation of dried [ 18 F]TBAF
The [ 18 F]F − aqueous was taken in a clean glass vial and Bu 4 NOAc solution (TBAA: 3.2 mg, 10.0 μmol; 500 μL CH 3 CN) was added to disperse [ 18 F]F − uniformly at the bottom of the vial.The solution was azeotropically dried for three times (300 μL anhydrous CH 3 CN × 3) at 100 °C with nitrogen flow.Then the reaction vial was capped to obtain dried [ 18 F]TBAF for radiolabeling.

The preparation of dried [ 18 F]CsF
The [ 18 F]F − aqueous was taken in a clean glass vial and CsOAc solution (CsOAc: 1.0 mg, 5.0 μmol; 200 μL H 2 O) was added to disperse [ 18 F]F − uniformly at the bottom of the vial.The solution was azeotropically dried for three times (300 μL anhydrous CH 3 CN × 3) at 100 °C with nitrogen flow.Then the reaction vial was capped to obtain dried [ 18 F]CsF for radiolabeling.

A m of [ 18 F]4
Solutions of 4 at graded concentrations were prepared and analyzed by an analytical HPLC shown in Additional file 1: Table S21 (phase A: CH3CN; phase B: ultrapure water; isocratic elution at 40% phase A and 60% phase B. Flow rate: 1.0 mL•min −1 , 20 min, UV = 254 nm.).The UV absorption peak areas at different amounts of 4 was measured, and the relationship between the areas of the absorption peaks and the amounts of the substance was obtained by linear analysis in Additional file 1: Figure S7.
The A m of [ 18 F]4 was calculated by dividing the radioactivity of [ 18 F]4 at end of synthesis by the molar amount of 4 measured by HPLC-UV [nmol of 4] as interpreted from the UV-standard curve.